The binding of digoxigenin-12d-H3 to NaK ATPase was studied directly at room temperature. Digoxigenin bound to the enzyme reversibly with high affinity only in the presence of ligands. Like cardiac glycoside binding, mixtures Mg2 ion and Pi or Na ion, Mg2 ion and ATP were both effective as ligands but such binding was eliminated by ouabain-pretreatment. Scatchard plots of the binding were linear and showed that the dissociation constant did not change at different concentration range, but the number of binding sites on the enzyme was reduced on decreasing the ligand concentration. These results show that the binding of digoxigenin to the NaK-ATPase does not follow the usual equation representing a reversible reaction. BIBLIOGRAPHIC REFERENCES: Yoda, S., Sarrif, A.M. and Yoda, A. Structure-activity relationships of cardiotonic steroids for the inhibition of sodium-and potassium-dependent adenosine triphosphatase IV. Dissociation rate constants for complexes of the enzyme with cardiac oligodigitoxides. Mol. Pharmacol. 11 647 (1975). Yoda, A. and Yoda, S. Structure-activity relationships of cardiotonic steroids for the inhibition of sodium-and potassium-dependent adenosine triphosphatase V. Dissociation rate constants of digitoxin acetates. Mol. Pharmacol. 11 653 (1975).